This invention relates to a process for the production of Streptovaricin C.
U.S. Pat. No. 3,116,202 describes streptovaricins and their production. As disclosed therein, Streptomyces spectabilis (NRRL2494) produces five types of streptovaricins designated as types A, B, C, D, and E. It also describes the use of streptovaricin as an antituberculosis drug. However, this product has not achieved broad use for this purpose.
Example 3 of this patent discloses the accumulation of 21 mg/l of streptovaricin in the broth assuming that the crude product was 100% pure. However, the disclosure gives no indication of the amount or ratio of Streptovaricin C, in the crude mixture.
More recent attempts have been made to obtain novel antibiotics by chemically modifying Streptovaricin C in order to provide anti-virus and anti-cancer agents (See K. Onodera et al., The Journal of Antibiotics, Feb. 1986, pp.147-154). (See K. Onodera et al., The Journal of Antibiotics, May 1989, pp. 779-787.)
These derivatives use only Streptovaricin C and thus require a method for selectively preparing Streptovaricin C. In the May 1989 K. Onodera et al article, the yield of Streptovaricin C, the most abundant component of the streptovaricin complex provided by the Upjohn Co. and used by the authors, was about 20%. (See pages 151-152).
In K. Rinehart et al., Biochemistry, Vol. 13, No. 5, 1974, pp. 861-867, the content of Streptovaricin C within the mixture of streptovaricins obtained from the provider (Upjohn 11560-3), was about 10 to 20%. This suggests that the Streptovaricin C content in the broth of Example 3 of U.S. Pat. No. 3,116,202 was about 2-4 mg/l. These amounts and concentrations are not sufficient for further development of Streptovaricin C derivatives.
A more efficient method for separating Streptovaricin C from a culture broth of a streptovaricin producing strain which produces a mixture of types A, B, C, D, and E, is disclosed in Japanese applications No. 14285/1990 and 14286/1990. (See also H. Wang, Annals New York Academy of Sciences, 431, 1983, pp.313-321.) In these methods, a publicly available streptovaricin-producing strain (ATCC 27465) is cultured in the presence of a non-ionic adsorbent and with the optional addition of fumaric acid or one of its water-soluble salts. Using these methods, it was possible to increase the amount of Streptovaricin C separated from the culture broth. Even so, higher productivity of streptovaricin is needed for proper development and commercial production of this compound.
U.S. patent applications Ser. Nos. 07/601,875 and 07/601,877, filed on Oct. 23, 1990, respectively, the contents of each of which are incorporated herein by reference, disclose methods for increasing the efficiency of producing and separating Streptovaricin C from a culture broth. This is achieved by carrying out the fermentation in the presence of a nonionic adsorbent, e.g., in the form of beads, and effecting separation of the beads from the fermentation broth by adjusting the specific gravity of the fermentation broth.
U.S. patent application Ser. No. 07/601,876, filed Oct. 23, 1990 now U.S. Pat. No. 5,114,846, the contents of which are incorporated herein by reference, discloses a method for selectively producing Streptovaricin C from a culture broth resulting in isolation of Streptovaricin C and a method for selecting a natural mutant strain belonging to the genus Streptomyces which is a hyper-producer of Streptovaricin C. The hyperproducing strain obtained by this method produced streptovaricin in an amount of about 600 to 700 mg/liter. This strain has been deposited at the Fermentation Institute in Japan under Deposit No. FERM BP-3460. The Streptovaricin C hyperproducing strains are selected from Streptomyces spectabilis by culturing Streptomyces spectabilis, and separating those colonies which are non-spore forming (asporogenous). The selected colonies are then separately cultured and tested for streptovaricin productivity. The colony or colonies having the highest desirable streptovaricin productivity is then fermented in a nutrient broth containing a compound selected from the group consisting of fumaric acid and water-soluble salts thereof, and adsorbent polymer beads and the streptovaricin produced are recovered in the usual manner.